Adrenal Zona Glomerulosa Cells Research Articles

PGE2 stimulates aldosterone release via cAMP/sPRR/β-catenin pathway in mouse inner medullary collecting duct cells

Increasing evidence showed the generation of aldosterone in the collecting duct (CD), a major site for the synthesis and action of prostaglandin E2 (PGE2). Both aldosterone and PGE2 are stimulated by Na+ depletion or K+ loading or angiotensin II. However, what happens first and the correlation between PGE2 and aldosterone in the CD remains obscure. PGE2 stimulates aldosterone release in the adrenal zona glomerulosa cells via E-prostanoid (EP) receptors by activating the cAMP/cAMP-responsive element-binding pathway. Here, we further tested the hypothesis that the synthesis of aldosterone is stimulated by PGE2 in CD cells and the underlying mechanism. In primary mouse inner medullary CD cells, PGE2 incubation significantly stimulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2), (pro)renin receptor (PRR), and prorenin protein expression and upregulation of medium soluble PRR (sPRR), prorenin/renin, and cAMP levels, both of which were attenuated by the EP1 antagonist SC-19220 and the protein kinase C (PKC) inhibitor calphostin C. PGE2-induced release of aldosterone, prorenin/renin, and sPRR was also abolished by the protein kinase A inhibitor H89. Both a PRR antagonist PRO20 and β-catenin inhibitor ICG001 blunted PGE2 or sPRR-induced aldosterone and prorenin/renin secretion. Exogenous sPRR protein incubation not only stimulated the release of aldosterone and prorenin/renin but also reversed PF429242 (an inhibitor of subtilisin kexin isozyme-1, which can block endogenous sPRR generation)-attenuated aldosterone and prorenin/renin secretion. Therefore, we conclude that PGE2 increases CD aldosterone synthesis through the EP1 receptor via the PKC/cAMP/sPRR/β-catenin pathway.

HEPARIN-INDUCED RENAL TUBULAR ACIDOSIS MASQUERADING AS HYPERKALEMIA IN A PATIENT WITH SARS-COV-2

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Renal tubular acidosis (RTA) is defined as inadequate excretion of acids via the kidneys to maintain the acid-base balance. In RTA type 4, there is either deficiency or resistance to aldosterone leading to impaired regulation of electrolytes and hydrogen ions. This leads to hyponatremia, hyperkalemia, and acidemia. We present an unsuspecting etiology to RTA type 4 in a critically ill. SARS-CoV-2 patient as noted by heparin use for VTE prophylaxis. [1,2] CASE PRESENTATION: A 72-year-old male with history of chronic kidney disease, leukemia in remission presents with 6-day history of flu-like symptoms including intermittent diarrhea, fevers, decreased appetite, shortness of breath and generalized malaise with exposure to several known sick contacts with SARS-CoV-2. The patient was admitted to the ICU for acute hypoxic respiratory failure and started on Decadron, Remdesivir, Azithromycin, Ceftriaxone, Lovenox, Zinc, and vitamin C and subsequently intubated on day 10. Creatinine up trended and peaked at 3.51 mg/dL on hospital day 14. He developed hyperkalemia initially presumed to be secondary to acute kidney injury. His platelet counts declined by >50%, >10 days after hospitalization. Due to concern for HIT, argatroban was started but stopped due to bleeding. As fondaparinux is contraindicated in renal failure, he was restarted on heparin when HIT ab (-) result was obtained. Serum potassium peaked at 6.7 mmol/L on day 21. He was treated with calcium gluconate, insulin D50, Patiromer 8.4 g with no significant improvement in potassium levels. Due to concerns that use of heparin may be causing hyperkalemia by suppressing aldosterone, heparin was discontinued. Subsequently, serum potassium levels improved to normal range. DISCUSSION: In RTA type 4, either low levels of aldosterone or from kidneys not responding to aldosterone, impaired sodium reabsorption and potassium secretion occurs.[2]. Persistent hyperkalemia in whom there is no apparent cause such as potassium supplements or a potassium-sparing diuretic or renal failure, hypoaldosteronism must be considered [3-5].In COVID-19, there exists a higher propensity for thromboembolic disease and often prevention with heparin can serve as the best treatment. Heparin has a direct toxic impact on the adrenal zona glomerulosa cells, which may be facilitated by a drop in the number and affinity of adrenal angiotensin II receptors [4,6]. This reduction in aldosterone can lead to severe hyperkalemia [6]. CONCLUSIONS: This case is unique because hyperkalemia was noted to be refractory to the typical standard treatment given the etiology was hypoaldosteronism likely secondary to heparin use. Most patients with RTA if treated adequately in the early stages do not develop permanent kidney failure, have better mortality and morbidity outcomes and necessitate a broad etiologic differential. REFERENCE #1: Rodriguez Soriano J. Renal tubular acidosis: the clinical entity. J Am Soc Nephrol 2002;13:2160. REFERENCE #2: Renal Tubular Acidosis. www.kidney.niddk.nih.gov.U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. National Institutes of Health, NIH Publication No. 09–4696, October 2008. REFERENCE #3: DeFronzo RA. Hyperkalemia and hyporeninemic hypoaldosteronism. Kidney Int 1980;17:118. DISCLOSURES: No relevant relationships by Vidya Baleguli, source=Web Response No relevant relationships by Martin Herrera, source=Web Response No relevant relationships by Riaz Mahmood, source=Web Response No relevant relationships by Erine Raybon-Rojas, source=Web Response

Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Calcineurin-NFATc4 Pathway Is Activated Upon K+-stimulation of Adrenal Aldosterone Production

The mineralocorticoid aldosterone secreted by the adrenal zona glomerulosa (ZG) cells promote renal K+ secretion and Na+ reabsorption; thereby it is critical for the control of ion homeostasis and blood pressure. While the Ca2+/calmodulin-dependent protein kinase (CAMK) pathway regulating K+ stimulated aldosterone production is well studied, little is known about the potentially involved phosphatases. Interestingly, immunosuppression therapy of transplanted patients with protein phosphatase 3 (calcineurin) inhibitors often results in rather low plasma aldosterone levels despite a concomitant hyperkalemia and hyperreninemia. Calcineurin (Cn) is a calcium and calmodulin-dependent protein phosphatase expressed in the adrenal cortex. We tested the hypothesis that Cn participates in the signal transduction pathway mediating the K+-dependent stimulation of aldosterone production. To address this question, we used the adrenocortical cell model NCI-H295R, mouse and human ex vivo adrenal preparations and a ZG-specific and inducible Cn knockout mouse model (ZG-CnB1-KO). Inhibition of Cn with tacrolimus abolished the K+-stimulated expression of CYP11B2 in NCI-H295R cell line as well as mouse and human adrenal pieces, ex vivo. Using a phosphoproteomics analysis, we identified nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a critical downstream factor mediating Cn function. In support of this result, genetic deletion of NFATc4 reduced the basal expression of CYP11B2 and impaired the K+-stimulated expression of this gene. Conversely, the expression of a constitutively active form of NFATc4 drastically increased the expression of CYP11B2 in NCI-H295R cells which remained unaltered upon treatment with K+ or tacrolimus. Finally, preliminary experiments using ZG-CnB1-KO mice suggest that Cn deletion in the ZG blunts the increase in aldosterone excretion triggered by high K+ diet. Altogether, our data indicate that Cn function is indispensable for the physiological regulation of aldosterone production. Moreover, Cn may represent a novel molecular target for the pharmacological treatment of primary aldosteronism.

L- and T-type calcium channels control aldosterone production from human adrenals.

Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenal adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type CaV3.2/3.3 and L-type CaV1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K+. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effect on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.

Role of SCTR/AT1aR heteromer in mediating ANGII-induced aldosterone secretion.

The involvement of secretin (SCT) and its receptor (SCTR) in angiotensin II (ANGII)-mediated osmoregulation by forming SCTR/ angiotensin II type 1 receptor (AT1R) heteromer is well established. In this study, we demonstrated that SCTR/AT1R complex can mediate ANGII-induced aldosterone secretion/release through potentiating calcium mobilization. Through IHC and cAMP studies, we showed the presence of functional SCTR and AT1R in the primary zona glomerulosa (ZG) cells of C57BL/6N (C57), and functional AT1R and non-functional SCTR in SCTR knockout (SCTR-/-) mice. Calcium mobilization studies revealed the important role of SCTR on ANGII-mediated calcium mobilization in adrenal gland. The fluo4-AM loaded primary adrenal ZG cells from the C57 mice displayed a dose-dependent increase in intracellular calcium influx ([Ca2+]i) when exposed to ANGII but not from the SCTR-/- ZG cells. Synthetic SCTR transmembrane (TM) peptides STM-II/-IV were able to alter [Ca2+]i in C57 mice, but not the mice with mutated STM-II/-IV (STM-IIm/IVm) peptides. Through enzyme immunoassay (EIA), we measured the aldosterone release from primary ZG cells of both C57 and SCTR-/- mice by exposing them to ANGII (10nM). SCTR-/- ZG cells showed impaired ANGII-induced aldosterone secretion compared to the C57 mice. TM peptide, STM-II hindered the aldosterone secretion in ZG cells of C57 mice. These findings support the involvement of SCTR/AT1R heterodimer complex in aldosterone secretion/release through [Ca2+]i.

Secretion of adrenal zona glomerulosa cells in rats exposed to low doses of dichlorodiphenyltrichloroethane during prenatal and postnatal development

Secretion of adrenal zona glomerulosa cells in rats exposed to low doses of dichlorodiphenyltrichloroethane during prenatal and postnatal development

Activation of Renin‐Angiotensin Aldosterone System's Effect on Protein Disulfide Isomerase

Disordered activation of the Renin‐Angiotensin‐Aldosterone‐System (RAAS) is associated with hypertension and insulin resistance. The hormones Angiotensin II (Ang II) and Aldosterone (Aldo) are two of its principal effector molecules that have been shown to regulate sodium and volume homeostasis leading to blood pressure control. Ang II has been shown to activate endothelial cells and increase the secretion of extracellular protein disulfide isomerase (PDI). PDI is a multifunctional protein critical to thrombus formation and regulation of reactive oxygen species among other functions. Our group has recently reported that caloric restriction (CR) in rodents leads to increases in Aldo secretion from adrenal zona glomerulosa cells. However, the effects of CR on PDI are unknown. We hypothesize that CR, due to its association with activating RAAS, will lead to increases in the circulating PDI activity. To test our hypothesis, we propose to study the in vitro effects of AngII and Aldo on PDI activity. We will study the in vivo effects of CR on the circulating activity of PDI in Long‐Evans Tokushima Fatty (LETO) and Otsuka Long‐Evans Tokushima Fatty (OLETF). We use two assays to measure PDI activity. In one approach we will use insulin degradation assays. The measurement of circulating PDI activity will be further validated with a fluorescent of Di‐E‐GSSG assay. Initial studies have shown specificity of our assay using the insulin degradation assay and recombinant PDI. Controls of our fluorescent assay are currently underway. We expect that CR will be associated with increased PDI activity.Support or Funding InformationK.V. is supported by an NIH MARC grant R25HL121029 and by the Division of Medical Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeted disruption of the Kcnj5 gene in the female mouse lowers aldosterone levels.

Aldosterone is released from adrenal zona glomerulosa (ZG) cells and plays an important role in Na and K homoeostasis. Mutations in the human inwardly rectifying K channel CNJ type (KCNJ) 5 (KCNJ5) gene encoding the G-coupled inwardly rectifying K channel 4 (GIRK4) cause abnormal aldosterone secretion and hypertension. To better understand the role of wild-type (WT) GIRK4 in regulating aldosterone release, we have looked at aldosterone secretion in a Kcnj5 knockout (KO) mouse. We found that female but not male KO mice have reduced aldosterone levels compared with WT female controls, but higher levels of aldosterone after angiotensin II (Ang-II) stimulation. These differences could not be explained by sex differences in aldosterone synthase (Cyp11B2) gene expression in the mouse adrenal. Using RNAseq analysis to compare WT and KO adrenals, we showed that females also have a much larger set of differentially expressed adrenal genes than males (395 compared with 7). Ingenuity Pathway Analysis (IPA) of this gene set suggested that peroxisome proliferator activated receptor (PPAR) nuclear receptors regulated aldosterone production and altered signalling in the female KO mouse, which could explain the reduced aldosterone secretion. We tested this hypothesis in H295R adrenal cells and showed that the selective PPARα agonist fenofibrate can stimulate aldosterone production and induce Cyp11b2. Dosing mice in vivo produced similar results. Together our data show that Kcnj5 is important for baseline aldosterone secretion, but its importance is sex-limited at least in the mouse. It also highlights a novel regulatory pathway for aldosterone secretion through PPARα that may have translational potential in human hyperaldosteronism.

Abstract 057: Cellular and Genetic Causes of Idiopathic Hyperaldosteronism

Background: Primary aldosteronism (PA) affects ~10% of hypertensive patients and has unilateral and bilateral forms (30%:70%). Unilateral PA is caused by aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone regulating genes ( KCNJ5>>CACNA1D ). We recently demonstrated that adrenals from normotensive patients present with pockets of cell expressing aldosterone synthase (CYP11B2). These aldosterone-producing foci (APF) have somatic gene mutations similar to those found in APA ( CACNA1D >> KCNJ5 ). Bilateral PA, which is typically treated by mineralocorticoid receptor (MR) blockade rather than surgery, is termed idiopathic hyperaldosteronism (IHA). Its pathobiology is largely unknown but has been thought to be due to zona glomerulosa (ZG) hyperplasia. Methods: We studied 11 IHA patients (7 males, 4 females) who had unilateral adrenalectomy. Immunohistochemistry for CYP11B2 and next generation sequencing (NGS) targeting genes found in APA were performed on formalin fixed paraffin embedded adrenal tissue. Results were compared to previously described cohorts of 53 age-matched normotensive patients (29 males, 24 females) which were evaluated similarly. Results: CYP11B2 expression was absent from intervening ZG cells in 8/11 (73%) IHA adrenals, but all adrenals harbored at least one APF. The median number and size of APF per case were significantly larger in IHA than normotensive controls (6.1 vs 0 APF/cm 2 of adrenal cortex and 0.25 vs. 0.16 mm 2 , respectively; p<0.0001 and p<0.006). In this IHA cohort, NGS identified CACNA1D and KCNJ5 somatic mutations in 44/71 (62%) and 1/71 (1%) of APF, respectively. Interpretations. Diffuse CYP11B2 expression in adrenal ZG cells was only observed in 3/11 IHA cases, arguing against ZG hyperplasia as the major underlying pathobiology. Rather, we demonstrated increased and enlarged APF in IHA adrenals compared to normotensive controls, supporting potential contribution to the clinical manifestations of hyperaldosteronism. The frequent occurrence of aldosterone-dysregulating CACNA1D somatic mutations in APF support CACNA1D as a potential therapeutic target in IHA to complement current MR blockade approaches.

245 - MANP: A Novel Aldosterone Inhibitor: Potential Molecular Mechanisms in Vitro in Human Adrenal Cells

Introduction: Aldosterone (Aldo) is synthesized and secreted by the adrenal cortex zona glomerulosa (ZG) cells. In heart failure (HF) patients Aldo is increased and plays a critical role in inducing and maintaining sodium and water retention as well as mediating myocardial remodeling and fibrosis. MANP is a novel Mayo Clinic designer natriuretic peptide (NP) which was demonstrated to inhibit circulating Aldo levels in vivo canines and in human subjects. Importantly, 3′, 5′ cyclic guanosine monophosphate (cGMP) generated by NPs activates protein kinase G (PKG) and also binds to phosphodiesterases (PDEs). Both PKG and PDEs have been suggested to play a role in the actions of NPs. To date no study has defined the direct cellular inhibitory effects of MANP on Aldo in human adrenal cells. Our goals were therefore to define the mechanisms of the inhibitory actions of MANP on Aldo in ZG cells with a special focus on cGMP and PDE's. Hypothesis: We hypothesized that MANP directly inhibits Aldo in adrenal cortex ZG cells and the inhibitory effect is mediated partially through PDEs. Methods: The ZG cell line H295R cells were cultured in 24-well plates for 24 hrs and starved overnight with serum free medium before stimulation/treatment. MANP (10−8, 10−7 or 10−6 M) was added to cells after starvation. Angiotensin II (ANG II, 10−8, 10−7 or 10−6 M) was employed to stimulate Aldo release. Supernatants were collected after 24 hrs for Aldo quantification with commercial ELISA kits. The PDE inhibitor IBMX (500 uM) and protein kinase G (PKG) specific cGMP analog 8-CPT-cGMP (100 uM) were used. Data are presented as Mean ± SEM, *P < .05 between two groups by unpaired t-test. Results: Angiotensin II stimulated Aldo secretion in a dose-dependent manner. MANP inhibited ANG II stimulated Aldo in a dose dependent manner (10−8 M MANP + ANG II 38.5 ± 2.0, 10−7 M MANP + ANG II 28.8 ± 1.8*, 10−6 M MANP + ANG II 28.0 ± 1.9* vs ANG II 40.5 ± 1.3 ng/dL). The PKG-specific analogue 8-CPT-cGMP surprisingly increased Aldo levels (50.7 ± 2.4* vs ANG II 34.9 ± 0.5 ng/dL). Non-specific PDEs inhibition with IBMX attenuated the inhibitory effect of MANP on Aldo (IBMX + MANP + ANG II 34.9 ± 0.6 vs MANP + ANG II 10−7 M 24.7 ± 2.5* vs ANG II 34.9 ± 0.5 ng/dL). Conclusion: We conclude that MANP directly inhibits Aldo in human ZG cells and the inhibition of Aldo is mediated in part through PDEs. The potent Aldo suppressing property of MANP may render it a promising new generation Aldo inhibiting drug for HF and other cardiovascular disease states of aldosterone excess.

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K+ channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.

Abstract 128: Leptin: A Regulator of Aldosterone Synthase Expression &amp; Aldosterone Secretion in Visceral Adipocytes, in Mice

Obesity is associated with inappropriately high aldosterone levels, which contribute to the development of metabolic and cardiovascular disorders. The origin of these high aldosterone levels is incompletely understood. We recently demonstrated that the adipocyte-derived hormone leptin regulates aldosterone synthase (CYP11B2) expression and stimulates aldosterone release from adrenal zona glomerulosa cells. Recent studies demonstrate that adipocytes express CYP11B2 and secrete aldosterone. However, the mechanisms regulating aldosterone release from adipocytes remain unclear. Likewise, whether visceral (Visc) and subcutaneous (SubQ) adipose tissue contribute to a similar extent to aldosterone production is unknown. We tested the hypothesis that leptin increases adipocyte CYP11B2 expression and aldosterone production and investigated whether Visc and SubQ adipose tissues respond similarly to leptin. Immunostaining of mouse adipose tissue cross-sections and isolated mature adipocytes revealed that Visc and SubQ adipose tissue express leptin receptors. Treatment of mouse freshly isolated mature adipocytes, non-differenciated (stromal fraction) and differentiated adipocytes revealed that leptin dose-dependently increased CYP11B2 expression and aldosterone production in Visc adipose tissue only. Although leptin receptor and CYP11B2 levels were similar in SubQ and Visc adipocytes, SubQ adipocytes were unresponsive to leptin. The physiological relevance of these in vitro data was tested by measuring plasma aldosterone levels in mice deprived of adipose tissue (lipodystrophic mice) treated with leptin. Absence of adipose tissue in lipodystrophic mice blunted leptin-induced increases in aldosterone levels (WT-vehicle: 471±82 vs. WT-Leptin: 1699±396, p&lt;0.05; KO-vehicle: 539±71 vs. KO+leptin: 787±156, NS). The human relevance of these data was determined by reporting that CYP11B2 expression gradually increased with body mass index in human mediastinal and omental fat depots. In summary these data strongly suggest that leptin regulates CYP11B2 levels and aldosterone release in visceral adipose tissue and that leptin-induced, adipocyte-derived aldosterone may contribute to obesity-associated hyperaldosteronism.

Abstract 097: Effects of Lysine-specific Demethylase 1 Deficiency on Aldosterone Production and Salt-sensitive Hypertension in Female Mice

Lysine-Specific Demethylase1 (LSD1) is an epigenetic factor modulated by salt intake. Previously, we documented the male heterozygote LSD1 knockout mice (LSD1+/-) had dysregulation of aldosterone (ALDO) production on a liberal salt diet (1.6% Na+) associated with salt-sensitive hypertension. This study assessed if: 1) female LSD1+/- mice have a similar phenotype; and 2) the effect of aging on this phenotype. Methods: Female LSD1+/- and wild type mice (LSD1+/+) were randomly assigned for sacrifice at the ages of 18-week, 52-week, and 75-week and the following were assessed at each time point: blood pressure (BP); plasma renin activity (PRA) and ALDO; urine albumin; and ex vivo ALDO production from isolated adrenal zona glomerulosa cells. Results: BP and urine albumin in the LSD1+/- compared to the LSD1+/+ were not different at any age (Table). However, the LSD1+/- had greater ALDO/PRA ratios at 18 weeks compared with the LSD1+/+, but lower ALDO levels and ex vivo ALDO production at 52 and 75 weeks. Associated with this phenotype, the LSD1+/- showed significantly higher rate of all-cause mortality than the LSD1+/+. Conclusion: Lack of LSD1 caused dysregulation of ALDO production in both male and female mice. But the cardiovascular outcomes are different. The LSD1+/- females in contrast to males do not develop hypertension or albuminuria even at 75 weeks of age. However, the females do die at a faster rate than the males of a variety of causes. Thus, there is considerable sexual dimorphism in the pathogenesis of cardiovascular outcomes associated with dysregulation of adrenal ALDO production mediated by lack of LSD1.

Does Aldosterone Play a Significant Role for Regulation of Vascular Tone?

Besides the well-known renal effects of aldosterone, the hormone is now known to have direct vascular effects. Clinical observations underline substantial adverse effects of aldosterone on cardiovascular function. The source of systemic circulating aldosterone is the adrenal gland zona glomerulosa cells through stimulus-secretion coupling involving depolarization, opening of L- and T-type calcium channels and aldosterone synthase activation. Local formation and release in peripheral tissues such as perivascular fat is recognized. Where does aldosterone affect the vasculature? Mineralocorticoid receptors (MRs) are present in endothelial and vascular smooth muscle cells, and MR-independent pathways are also involved. The vascular effects of aldosterone are complex, both concentration and temporal and spatial aspects are relevant. The acute response includes vasodilation through endothelial nitric oxide formation and vasoconstrictor effects through endothelial-contracting cyclooxygenase-derived factors and a changed calcium handling. The response to aldosterone can change within the same blood vessels depending on the exposure time and status of the endothelium. Chronic responses involve changed levels of reactive oxygen radicals, endothelial Na-influx and smooth muscle calcium channel expression. Furthermore, perivascular cells for example mast cells have also been suggested to participate in the chronic response. Moreover, the vascular effect of aldosterone depends on the status of the endothelium which is likely the cause of the very different responses to aldosterone and MR treatment observed in human studies going from increased to decreased flow depending on whether the patient had prior cardiovascular disease with endothelial dysfunction or not. A preponderance of constrictor versus dilator responses to aldosterone could therefore be involved in the detrimental vascular actions of the hormone in the setting of endothelial dysfunction and contribute to explain the beneficial action of MR blockers on blood pressure and target organ injury.

Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis.

Deficiency of the antiaging gene Klotho (KL) induces renal damage and hypertension through unknown mechanisms. In this study, we assessed whether KL regulates expression of CYP11B2, a key rate-limiting enzyme in aldosterone synthesis, in adrenal glands. We found that haplodeficiency of KL(+/-) in mice increased the plasma level of aldosterone by 16 weeks of age, which coincided with spontaneous and persistent elevation of BP. Blockade of aldosterone actions by eplerenone reversed KL deficiency-induced hypertension and attenuated the kidney damage. Protein expression of CYP11B2 was upregulated in adrenal cortex of KL(+/-) mice. KL and CYP11B2 proteins colocalized in adrenal zona glomerulosa cells. Silencing of KL upregulated and overexpression of KL downregulated CYP11B2 expression in human adrenocortical cells. Notably, silencing of KL decreased expression of SF-1, a negative transcription factor of CYP11B2, but increased phosphorylation of ATF2, a positive transcription factor of CYP11B2, which may contribute to upregulation of CYP11B2 expression. Therefore, these results show that KL regulates adrenal CYP11B2 expression. KL deficiency-induced spontaneous hypertension and kidney damage may be partially attributed to the upregulation of CYP11B2 expression and aldosterone synthesis.

Channels and Pumps in Aldosterone-Producing Adenomas

Primary aldosteronism is the most common cause of secondary hypertension, with a prevalence of about 5% of the general hypertensive population and 10% of patients referred to hypertension specialty clinics. Aldosterone-producing adenomas (APAs) account for approximately 30–50% of cases of primary aldosteronism, and idiopathic hyperaldosteronism accounts for most of the rest of the cases. Aldosterone synthesis is regulated primarily by the renin-angiotensin system, potassium, ACTH, and to a lesser degree other peptides (1). Isolated adrenal zona glomerulosa and fasciculata cells from different species maintain a negative membrane resting potential determined by regulating membrane permeability to potassium (2, 3). Zona glomerulosa cells with hyperpolarized resting membrane voltages are steroidogenically inactive (4). Stimulation with angiotensin II, ACTH, or potassium results in membrane depolarization, opening of plasma membrane calcium channels, and mobilization of intracellular calcium, which increases activation of the calcium-calmodulin pathway, ultimately resulting in an increase in steroid biosynthesis and secretion (1). Several potassium channels have been described in rodent, bovine, and human adrenal cells, including inward rectifying and leak potassium currents that provide background potassium currents (reviewed in Ref. 3).

Minireview: Potassium Channels and Aldosterone Dysregulation: Is Primary Aldosteronism a Potassium Channelopathy?

Primary aldosteronism is the most common form of secondary hypertension and has significant cardiovascular consequences. Aldosterone-producing adenomas (APAs) are responsible for half the cases of primary aldosteronism, and about half have mutations of the G protein-activated inward rectifying potassium channel Kir3.4. Under basal conditions, the adrenal zona glomerulosa cells are hyperpolarized with negative resting potentials determined by membrane permeability to K(+) mediated through various K(+) channels, including the leak K(+) channels TASK-1, TASK-3, and Twik-Related Potassium Channel 1, and G protein inward rectifying potassium channel Kir3.4. Angiotensin II decreases the activity of the leak K(+) channels and Kir3.4 channel and decreases the expression of the Kir3.4 channel, resulting in membrane depolarization, increased intracellular calcium, calcium-calmodulin pathway activation, and increased expression of cytochrome P450 aldosterone synthase (CYP11B2), the last enzyme for aldosterone production. Somatic mutations of the selectivity filter of the Kir3.4 channel in APA results in loss of selectivity for K(+) and entry of sodium, resulting in membrane depolarization, calcium mobilization, increased CYP11B2 expression, and hyperaldosteronism. Germ cell mutations cause familial hyperaldosteronism type 3, which is associated with adrenal zona glomerulosa hyperplasia, rather than adenoma. Less commonly, somatic mutations of the sodium-potassium ATPase, calcium ATPase, or the calcium channel calcium channel voltage-dependent L type alpha 1D have been found in some APAs. The regulation of aldosterone secretion is exerted to a significant degree by activation of membrane K(+) and calcium channels or pumps, so it is not surprising that the known causes of disorders of aldosterone secretion in APA have been channelopathies, which activate mechanisms that increase aldosterone synthesis.

Effects of Propylthiouracil on the Production of Aldosterone in Rat Zona Glomerulosa Cells

Propylthiouracil (PTU), an anti-thyroid drug, is widely used for the treatment in hyperthyroid patients. Previous studies indicate that thyroxine inhibit the spontaneous and adrenocorticotropin (ACTH)-stimulated corticosterone secretion by acting directly at adrenal glands via decrease cAMP production. It has also been shown that PTU suppresses corticosterone release by male rat zona fasciculata-reticularis cells. However, the direct effects and mechanisms of PTU on the aldosterone production in adrenal zona glomerulosa (ZG) cells are still unclear. ZG cells were prepared from adrenocortical tissues of male rats, and then challenged with or without angiotensin II (10^(-7), 10^(-6) M), ACTH (10^(-9) M), A23187 (10^(-5) M), cyclopiazonic acid (CPA, 10^(-5) M), forskolin (10^(-5) M), 8-Br-cAMP (10^(-5) M), trilostane (10^(-6) M, 3β-HSD inhibitor) or steroidogenic precursors (e.g. 10^(-6) M 25-OH-cholesterol and 10^(-7), 10^(-5) M corticosterone) at 37°C for 1 h. Our results showed that PTU (0-3 mM) dose-dependently decreased the aldosterone release in response to the above hormones, drugs and the steroidogenic precursors in vitro. PTU also decreased steroidogenic acute regulatory (StAR) protein expression, but did not alter the protein expression of P450 side chain cleavage enzyme (P450scc) during steroidogenesis of aldosterone. These results suggested that PTU has a direct inhibitory effect on aldosterone production via cAMP, and Ca^2+ down stream pathway and the steroidogenic enzymes activities including P450scc and aldosterone synthase, as well as StAR protein expression.

Effects of Propylthiouracil on the Production of Aldosterone in Rat Zona Glomerulosa Cells

Propylthiouracil (PTU), an anti-thyroid drug, is widely used for the treatment in hyperthyroid patients. Previous studies indicate that thyroxine inhibit the spontaneous and adrenocorticotropin (ACTH)-stimulated corticosterone secretion by acting directly at adrenal glands via decrease cAMP production. It has also been shown that PTU suppresses corticosterone release by male rat zona fasciculata-reticularis cells. However, the direct effects and mechanisms of PTU on the aldosterone production in adrenal zona glomerulosa (ZG) cells are still unclear. ZG cells were prepared from adrenocortical tissues of male rats, and then challenged with or without angiotensin II (10^(-7), 10^(-6) M), ACTH (10^(-9) M), A23187 (10^(-5) M), cyclopiazonic acid (CPA, 10^(-5) M), forskolin (10^(-5) M), 8-Br-cAMP (10^(-5) M), trilostane (10^(-6) M, 3β-HSD inhibitor) or steroidogenic precursors (e.g. 10^(-6) M 25-OH-cholesterol and 10^(-7), 10^(-5) M corticosterone) at 37°C for 1 h. Our results showed that PTU (0-3 mM) dose-dependently decreased the aldosterone release in response to the above hormones, drugs and the steroidogenic precursors in vitro. PTU also decreased steroidogenic acute regulatory (StAR) protein expression, but did not alter the protein expression of P450 side chain cleavage enzyme (P450scc) during steroidogenesis of aldosterone. These results suggested that PTU has a direct inhibitory effect on aldosterone production via cAMP, and Ca^2+ down stream pathway and the steroidogenic enzymes activities including P450scc and aldosterone synthase, as well as StAR protein expression.